US8889657B2 - Nanoparticle PEG modification with H-phosphonates - Google Patents

Nanoparticle PEG modification with H-phosphonates Download PDF

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US8889657B2
US8889657B2 US13/599,819 US201213599819A US8889657B2 US 8889657 B2 US8889657 B2 US 8889657B2 US 201213599819 A US201213599819 A US 201213599819A US 8889657 B2 US8889657 B2 US 8889657B2
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phosphonate
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Thomas E. Rogers
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Mallinckrodt LLC
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    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
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    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/40—Esters thereof
    • C07F9/4071—Esters thereof the ester moiety containing a substituent or a structure which is considered as characteristic
    • C—CHEMISTRY; METALLURGY
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
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    • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
    • C07J41/0055—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
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Definitions

  • nanoparticles can be rejected by a patient's immune system and processed before the nanoparticle can reach a target for delivery of a drug.
  • surfaces of nanoparticles can be modified.
  • nanoparticles such as liposomes
  • PEG polyethylene glycol
  • One approach includes combining lipid-PEG molecules to form liposomal formulations under mechanical means, e.g., extrusion.
  • liposomes can be preassembled and subsequently treated with a properly disposed PEG-lipid under conditions where the lipid can insert in the liposome bilayer, thereby forming a liposome with PEG attached to the liposome surface.
  • the present invention provides phosphonate conjugates and methods of preparing the phosphonate conjugates so as to allow, for example, improved methods and compounds for modifying the surface of a nanoparticle to increase in vivo circulation times and targeted delivery performance.
  • the phosphonate conjugates of the present invention can include a compound of the formula:
  • the present invention includes a compound of the formula:
  • the present invention includes a method of preparing a phosphonate conjugate, the method comprising: combining a primary amine compound having the formula: H 2 N(L 1 )-(R 1 ), a carbonyl compound having the formula: O ⁇ C[(L 2 )-(R 2 )] n , and a H-phosphonate compound having the formula:
  • the present invention includes a method of preparing a phosphonate conjugate, the method comprising: combining a secondary amine compound having the formula: HN[(L 1 )-(R 1 )](R 9 ), a carbonyl compound having the formula: O ⁇ C[(L 2 )-(R 2 )] n , and a H-phosphonate compound having the formula:
  • the phosphonate conjugates of the present invention and their methods of making provide a number of unique aspects to the areas of drug delivery and diagnostic imaging.
  • the present invention provides robust and simple methods for making compounds that can facilitate transforming nanoparticles into stealth nanoparticles that can be used for targeted drug delivery.
  • the phosphonate conjugates provide unique capability to increase the density of stealth agents on the surface of a nanoparticle and can, therefore, allow for additional flexibility in using different stealth agents that may not be as effective at lower densities.
  • the phosphonate conjugates can be produced in a fashion to provide additional stability to present stealth agents or other components, e.g., targeting agents, on the surface of a nanoparticle.
  • the phosphonate conjugates can incorporate positively- and/or negatively-charged groups to facilitate modification of the surface characteristics of nanoparticles.
  • the flexibility in making modified nanoparticles can, also, allow for tailored nanoparticles for specific therapeutic and/or diagnostic applications that can have long in vivo half-lives after administration to a patient.
  • FIG. 1 depicts an example synthetic method for producing a phosphonate conjugate of the present invention.
  • FIG. 2 depicts an example synthetic method for producing a phosphonate conjugate of the present invention.
  • FIG. 3 depicts an example synthetic method for making a phosphonate conjugate of the present invention.
  • FIG. 4 depicts an example synthetic method for making a phosphonate conjugate of the present invention on the surface of a liposome.
  • FIG. 5 depicts an example synthetic method for making a phosphonate conjugate of the present invention on the surface of a liposome.
  • FIG. 6 shows a general reaction scheme to combine diphenyl phosphite with a 1-alkanol to form the dialkyl hydrogen phosphonates, in accordance with exemplary embodiments of the present invention.
  • FIG. 7 shows a general reaction scheme for preparing a conjugate in accordance with an exemplary embodiment of the present invention.
  • FIG. 8 shows another general reaction scheme for preparing a conjugate in accordance with an exemplary embodiment of the present invention.
  • alkyl by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain hydrocarbon radical, having the number of carbon atoms designated (i.e., C 10-24 means ten to twenty-four carbons). In some embodiments, alkyl groups can range from one to thirty-six carbons. In certain embodiments, alkyl groups can range from one to ten carbons or one to twenty carbons. In some embodiments, the alkyl groups can be saturated or unsaturated, as well as substituted or unsubstituted.
  • substituted refers to a group that is bonded to a parent molecule or group.
  • an alkyl group having a cyano substituent is a cyano-substituted alkyl group.
  • Suitable substituents include, but are not limited to, halo, cyano, alkyl, amino, hydroxy, alkoxy, and amido.
  • H-phosphonate compound refers to compounds having the general formula of H—P(O)(OL 3 -R 3 )(OL 4 -R 4 ) and is further described herein.
  • primary amine compound refers generally to compounds having the general formula H 2 N(L 1 )-(R 1 ) and is further described herein.
  • second amine compound refers generally to compounds having the general formula HN[(L 1 )-(R 1 )](R 9 ) and is further described herein.
  • carbonyl compound refers generally to compounds having the general formula O ⁇ C[(L 2 )-(R 2 )] n and is further described herein.
  • the term “targeted delivery composition” refers to a composition of a nanoparticle attached to a phosphonate conjugate of the present invention, the specifics of which are described further herein.
  • the compositions of the present invention can be used as therapeutic compositions, as diagnostic compositions, or as both therapeutic and diagnostic compositions.
  • the compositions can be targeted to a specific target within a subject or a test sample, as described further herein.
  • nanoparticle refers to particles of varied size, shape, type and use, which are further described herein.
  • the characteristics of the nanoparticles can depend on the type and/or use of the nanoparticle as well as other factors generally well known in the art.
  • nanoparticles can range in size from about 1 nm to about 1000 nm. In other embodiments, nanoparticles can range in size from about 10 nm to about 200 nm. In yet other embodiments, nanoparticles can range in size from about 50 nm to about 150 nm.
  • the nanoparticles are greater in size than the renal excretion limit, e.g., greater than about 6 nm in diameter. In other embodiments, the nanoparticles are small enough to avoid clearance from the bloodstream by the liver, e.g., smaller than 1000 nm in diameter.
  • Nanoparticles can include spheres, cones, spheroids, and other shapes generally known in the art. Nanoparticles can be hollow (e.g., solid outer core with a hollow inner core) or solid or be multilayered with hollow and solid layers or a variety of solid layers. For example, a nanoparticle can include a solid core region and a solid outer encapsulating region, both of which can be cross-linked.
  • Nanoparticles can be composed of one substance or any combination of a variety of substances, including lipids, polymers, magnetic materials, or metallic materials, such as silica, gold, iron oxide, and the like.
  • Lipids can include fats, waxes, sterols, cholesterol, fat-soluble vitamins, monoglycerides, diglycerides, phospholipids, sphingolipids, glycolipids, cationic or anionic lipids, derivatized lipids, cardiolipin and the like.
  • Polymers can include block copolymers generally, poly(lactic acid), poly(lactic-co-glycolic acid), polyethylene glycol, acrylic polymers, cationic polymers, as well as other polymers known in the art for use in making nanoparticles.
  • the polymers can be biodegradable and/or biocompatible.
  • Nanoparticles can include a liposome, a micelle, a lipoprotein, a lipid-coated bubble, a block copolymer micelle, a polymersome, a niosome, a quantum dot, an iron oxide particle, a gold particle, a dendrimer, or a silica particle.
  • a lipid monolayer or bilayer can fully or partially coat a nanoparticle composed of a material capable of being coated by lipids, e.g., polymer nanoparticles.
  • liposomes can include multilamellar vesicles (MLV), large unilamellar vesicles (LUV), and small unilamellar vesicles (SUV).
  • the term “therapeutic agent” refers to a compound or molecule that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.
  • the present invention contemplates a broad range of therapeutic agents and their use in conjunction with the nanoparticles and phosphonate conjugates, as further described herein.
  • diagnostic agent refers to a component that can be detected in a subject or test sample and is further described herein.
  • linking group refers to part of a phosphonate conjugate that links portions of the conjugate.
  • a linking group, L 3 can link R 3 (e.g., an attachment component) to an oxygen bound to the phosphorous of the phosphonate conjugate.
  • R 3 e.g., an attachment component
  • the linking group can be assembled from readily available monomeric components to achieve an appropriate separation of targeting agent and other portions of a phosphonate conjugate that may, e.g., be attached to a nanoparticle.
  • a targeting agent refers to a molecule that is specific for a target.
  • a targeting agent can include a small molecule mimic of a target ligand (e.g., a peptide mimetic ligand), a target ligand (e.g., an RGD peptide containing peptide or folate amide), or an antibody or antibody fragment specific for a particular target.
  • Targeting agents can bind a wide variety of targets, including targets in organs, tissues, cells, extracellular matrix components, and/or intracellular compartments that can be associated with a specific developmental stage of a disease.
  • targets can include cancer cells, particularly cancer stem cells.
  • Targets can further include antigens on a surface of a cell, or a tumor marker that is an antigen present or more prevalent on a cancer cell as compared to normal tissue.
  • a targeting agent can further include folic acid derivatives, B-12 derivatives, integrin RGD peptides, RGD mimetics (Temming, K. et al., Drug Resistance Updates 8:381-402 (2005); Liu, S., Mol. Pharm. 3(5):472-87 (2006)), NGR derivatives, somatostatin derivatives or peptides that bind to the somatostatin receptor, e.g., octreotide and octreotate, and the like.
  • a targeting agent can be an aptamer—which is composed of nucleic acids (e.g., DNA or RNA), or a peptide and which binds to a specific target.
  • a targeting agent can be designed to bind specifically or non-specifically to receptor targets, particularly receptor targets that are expressed in association with tumors. Examples of receptor targets include, but are not limited to, MUC-1, EGFR, Claudin 4, MUC-4, CXCR4, CCR7, FOL1R, somatostatin receptor 4, Erb-B2 (erythroblastic leukaemia oncogene homologue 2) receptor, CD44 receptor, and VEGF receptor-2 kinase.
  • stealth agent refers to a molecule that can modify the surface properties of a nanoparticle and is further described herein.
  • tetrapodal presentation component refers to a molecule having the general formula:
  • each of L 5 and L 6 is independently selected from the group consisting of a bond and a linking group; and each R 5 and R 6 is independently selected from the group consisting of an C 1 -C 36 alkyl, a targeting agent, a diagnostic agent, and a stealth agent.
  • R 5 and/or R 6 can be hydrogen.
  • the term “embedded in” refers to the location of an agent on or in the vicinity of the surface of a nanoparticle.
  • Agents embedded in a nanoparticle can, for example, be located within a bilayer membrane of a liposome or located within an outer polymer shell of a nanoparticle so as to be contained within that shell.
  • the term “encapsulated in” refers to the location of an agent that is enclosed or completely contained within the inside of a nanoparticle.
  • therapeutic and/or diagnostic agents can be encapsulated so as to be present in the aqueous interior of the liposome. Release of such encapsulated agents can then be triggered by certain conditions intended to destabilize the liposome or otherwise effect release of the encapsulated agents.
  • an attachment component can be tethered to a nanoparticle so as to freely move about in solution surrounding the nanoparticle.
  • an attachment component can be tethered to the surface of a nanoparticle, extending away from the surface.
  • lipid refers to lipid molecules that can include fats, waxes, sterols, cholesterol, cholesterol derivatives, fat-soluble vitamins, monoglycerides, diglycerides, C 8 -C 36 alkyl, phospholipids, sphingolipids, glycolipids, cationic or anionic lipids, derivatized lipids, and the like.
  • Lipids can form micelles, monolayers, and bilayer membranes.
  • the lipids can self-assemble into liposomes.
  • the lipids can coat a surface of a nanoparticle as a monolayer or a bilayer.
  • aptamer refers to a non-naturally occurring oligonucleotide (typically 20-200 nucleotides) that specifically binds to a particular target. “Non-naturally occurring” encompasses non-naturally occurring sequences of natural nucleotides (A, T, C, G, U), as well as oligonucleotides with non-naturally occurring or modified nucleotides.
  • “Spiegelmers®” are aptamers with mirror image nucleic acids, i.e., in the L chiral configuration instead of the naturally occurring D configuration.
  • Aptamers can form unique three-dimensional structures via intramolecular interactions, and/or change structure upon binding to a target, e.g., via an induced-fit mechanism from a primary or secondary structure.
  • Aptamer binding to the target is not mediated by traditional complementary nucleic acid hybridization, e.g., double or triple helix formation, though portions of the aptamer may participate in such hybridization.
  • aptamers commonly form intramolecular hairpin structures and other three dimensional structures.
  • Aptamers can be selected according to any method or combination of methods.
  • Systematic Evolution of Ligands by Exponential Enrichment (SELEXTM), or a variation thereof, is commonly used in the field.
  • the basic SELEXTM process is described e.g., in U.S. Pat. No. 5,567,588. A number of variations on the basic method can also be used, e.g., in vivo SELEXTM, as described in US Appl. No. 2010015041.
  • MONOLEXTM is another selection process described, e.g., in Nitsche et al. (2007) BMC Biotechnology 7:48 and WO02/29093. In vivo selection using nucleic acid libraries injected into tumor cells is also possible (see, e.g., Mi et al., (2010) Nat. Chem. Biol. 1:22).
  • Aptamers for use in the present invention can be designed to bind to a variety of targets, including but not limited to MUC-1, EGFR, Claudin 4, MUC-4, CXCR4, CCR7, FOL1R, somatostatin receptor 4, Erb-B2 (erythroblastic leukaemia oncogene homologue 2) receptor, CD44 receptor, VEGF receptor-2 kinase, and nucleolin.
  • targets including but not limited to MUC-1, EGFR, Claudin 4, MUC-4, CXCR4, CCR7, FOL1R, somatostatin receptor 4, Erb-B2 (erythroblastic leukaemia oncogene homologue 2) receptor, CD44 receptor, VEGF receptor-2 kinase, and nucleolin.
  • subject refers to any mammal, in particular human, at any stage of life.
  • the terms “administer,” “administered,” or “administering” refers to methods of administering the targeted delivery compositions of the present invention.
  • the targeted delivery compositions of the present invention can be administered in a variety of ways, including topically, parenterally, intravenously, intradermally, intramuscularly, colonically, rectally or intraperitoneally. Parenteral administration and intravenous administration are the preferred methods of administration.
  • the targeted delivery compositions can also be administered as part of a composition or formulation.
  • the terms “treating” or “treatment” of a condition, disease, disorder, or syndrome includes (i) inhibiting the disease, disorder, or syndrome, i.e., arresting its development; and (ii) relieving the disease, disorder, or syndrome, i.e., causing regression of the disease, disorder, or syndrome.
  • inhibiting the disease, disorder, or syndrome i.e., arresting its development
  • relieving the disease, disorder, or syndrome i.e., causing regression of the disease, disorder, or syndrome.
  • adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by one of ordinary skill in the art.
  • formulation refers to a mixture of components for administration to a subject.
  • parenteral administration such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • Injection solutions and suspensions can also be prepared from sterile powders, granules, and tablets.
  • the formulations of a targeted delivery composition can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials.
  • a targeted delivery composition alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be “nebulized”) to be administered via inhalation through the mouth or the nose. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • Suitable formulations for rectal administration include, for example, suppositories, which comprises an effective amount of a targeted delivery composition with a suppository base.
  • Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons.
  • gelatin rectal capsules which contain a combination of the targeted delivery composition with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.
  • formulations can be administered topically or in the form of eye drops.
  • the phosphonate conjugates of the present invention and their methods of making provide a number of unique aspects to the areas of drug delivery and diagnostic imaging.
  • the present invention provides robust and simple methods for making compounds that can facilitate transforming nanoparticles into stealth nanoparticles that can be used for targeted drug delivery.
  • the phosphonate conjugates provide unique capability to increase the density of stealth agents on the surface of a nanoparticle and can, therefore, allow for additional flexibility in using different stealth agents that may not be as effective at lower densities.
  • the phosphonate conjugates can be produced in a fashion to provide additional stability to present stealth agents or other components, e.g., targeting agents, on the surface of a nanoparticle.
  • the phosphonate compounds can be integrated into nanoparticles by a variety of ways.
  • a H-phosphonate compound can be produced using methods described herein and then integrated with a nanoparticle such that the H-phosphonate compound is on the surface of the nanoparticle.
  • the H-phosphonate compounds can, for example, be integrated with a nanoparticle by incorporating the H-phosphonate compounds prior to making the nanoparticle.
  • a nanoparticle can be produced and the H-phosphonate compounds can be attached to the surface of the nanoparticle, thereby modifying the surface of the nanoparticle.
  • the flexibility in making modified nanoparticles can, for example, allow for tailored nanoparticles for specific therapeutic and/or diagnostic applications that can also have long in vivo half-lives after administration to a patient.
  • the compounds of the present invention can include a compound of the formula:
  • each L 1 , L 2 , L 3 and L 4 is independently selected from the group consisting of a bond and a linking group;
  • R 1 is selected from the group consisting of a nanoparticle, an attachment component, a targeting agent, a diagnostic agent, a stealth agent, and a tetrapodal presentation component;
  • each R 2 is independently selected from the group consisting of a stealth agent, C 1 -C 10 alkyl, a carboxylic acid or ester, a phosphonic acid or ester, a sulfonic acid or ester, and a hydroxy;
  • each R 3 and R 4 is independently selected from the group consisting of H, a nanoparticle, an attachment component, a targeting agent, a diagnostic agent, and a stealth agent, wherein at least one of R 3 or R 4 is other than H; and
  • n is an integer of from 0 to 2.
  • the compounds of the present invention can include a compound of the formula:
  • each L 1 , L 2 , L 3 and L 4 is independently selected from the group consisting of a bond and a linking group;
  • R 1 is selected from the group consisting of a nanoparticle, an attachment component, a targeting agent, a diagnostic agent and a stealth agent;
  • R 2 is independently selected from the group consisting of a stealth agent, C 1 -C 10 alkyl, a carboxylic acid or ester, a phosphonic acid or ester, a sulfonic acid or ester, and a hydroxy;
  • each R 3 and R 4 is independently selected from the group consisting of H, a nanoparticle, an attachment component, a targeting agent, a diagnostic agent, and stealth agent, wherein at least one of R 3 or R 4 is other than H;
  • R 9 is a C 1 -C 10 alkyl group; and
  • n is an integer from 0 to 2.
  • nanoparticles can be used in the present invention.
  • the characteristics of the nanoparticles can depend on the type and/or use of the nanoparticle as well as other factors generally well known in the art.
  • Suitable particles can be spheres, spheroids, flat, plate-shaped, tubes, cubes, cuboids, ovals, ellipses, cylinders, cones, or pyramids.
  • Suitable nanoparticles can range in size of greatest dimension (e.g., diameter) from about 1 nm to about 1000 nm, from about 10 nm to about 200 nm, and from about 50 nm to about 150 nm.
  • Nanoparticles can be made of a variety of materials generally known in the art.
  • nanoparticles can include one substance or any combination of a variety of substances, including lipids, polymers, or metallic materials, such as silica, gold, iron oxide, and the like.
  • examples of nanoparticles can include but are not limited to a liposome, a micelle, a lipoprotein, a lipid-coated bubble, a block copolymer micelle, a polymersome, a niosome, an iron oxide particle, a gold particle, a silica particle, a dendrimer, or a quantum dot.
  • the nanoparticles are liposomes composed partially or wholly of saturated or unsaturated lipids.
  • Suitable lipids can include but are not limited to fats, waxes, sterols, cholesterol, cholesterol derivatives, fat-soluble vitamins, monoglycerides, diglycerides, phospholipids, sphingolipids, glycolipids, derivatized lipids, and the like.
  • suitable lipids can include amphipathic, neutral, non-cationic, anionic, cationic, or hydrophobic lipids.
  • lipids can include those typically present in cellular membranes, such as phospholipids and/or sphingolipids.
  • Suitable phospholipids include but are not limited to phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), and phosphatidylinositol (PI).
  • Suitable sphingolipids include but are not limited to sphingosine, ceramide, sphingomyelin, cerebrosides, sulfatides, gangliosides, and phytosphingosine.
  • Other suitable lipids can include lipid extracts, such as egg PC, heart extract, brain extract, liver extract, and soy PC.
  • soy PC can include Hydro Soy PC (HSPC).
  • Cationic lipids include but are not limited to N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), and N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA).
  • DODAC N,N-dioleoyl-N,N-dimethylammonium chloride
  • DDAB N,N-distearyl-N,N-dimethylammonium bromide
  • DOTAP N-(1-(2,3-dioleoyloxy)propy
  • Non-cationic lipids include but are not limited to dimyristoyl phosphatidyl choline (DMPC), distearoyl phosphatidyl choline (DSPC), dioleoyl phosphatidyl choline (DOPC), dipalmitoyl phosphatidyl choline (DPPC), dimyristoyl phosphatidyl glycerol (DMPG), distearoyl phosphatidyl glycerol (DSPG), dioleoyl phosphatidyl glycerol (DOPG), dipalmitoyl phosphatidyl glycerol (DPPG), dimyristoyl phosphatidyl serine (DMPS), distearoyl phosphatidyl serine (DSPS), dioleoyl phosphatidyl serine (DOPS), dipalmitoyl phosphatidyl serine (DPPS), dioleoyl phosphatidyl ethanolamine
  • the lipids can include derivatized lipids, such as PEGlyated lipids.
  • Derivatized lipids can include, for example, DSPE-PEG 2000 , cholesterol-PEG 2000 , DSPE-polyglycerol, or other derivatives generally well known in the art.
  • lipids can be used to construct a nanoparticle, such as a liposome.
  • the lipid composition of a liposome can be tailored to affect characteristics of the liposomes, such as leakage rates, stability, particle size, zeta potential, protein binding, in vivo circulation, and/or accumulation in tissue, such as a tumor, liver, spleen or the like.
  • DSPC and/or cholesterol can be used to decrease leakage from the liposomes.
  • Negatively or positively lipids, such as DSPG and/or DOTAP can be included to affect the surface charge of a liposome.
  • the liposomes can include about ten or fewer types of lipids, or about five or fewer types of lipids, or about three or fewer types of lipids.
  • the molar percentage (mol %) of a specific type of lipid present typically comprises from about 0% to about 10%, from about 10% to about 30%, from about 30% to about 50%, from about 50% to about 70%, from about 70% to about 90%, from about 90% to 100% of the total lipid present in a nanoparticle, such as a liposome.
  • the lipids described herein can be included in a liposome, or the lipids can be used to coat a nanoparticle of the invention, such as a polymer nanoparticle. Coatings can be partially or wholly surrounding a nanoparticle and can include monolayers and/or bilayers.
  • a portion or all of a nanoparticle can include a polymer, such as a block copolymer or other polymers known in the art for making nanoparticles.
  • the polymers can be biodegradable and/or biocompatible.
  • Suitable polymers can include but are not limited to polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, and combinations thereof.
  • exemplary particles can include shell cross-linked knedels, which are further described in the following references: Becker et al., U.S. application Ser. No. 11/250,830; Thurmond, K.
  • suitable particles can include poly(lactic co-glycolic acid) (PLGA) (Fu, K. et al., Pharm Res., 27:100-106 (2000).
  • the nanoparticles can be partially or wholly composed of materials that are metallic in nature, such as silica, gold, iron oxide, and the like.
  • the silica particles can be hollow, porous, and/or mesoporous (Slowing, I. I., et al., Adv. Drug Deliv. Rev., 60 (11):1278-1288 (2008)).
  • Gold particles are generally known in the art, as provided by the following exemplary reference: Bhattacharya, R. & Mukherjee, P., Adv. Drug Deliv. Rev., 60(11): 1289-1306 (2008)).
  • Iron oxide particles or quantum dots can also be used and are well-known in the art (van Vlerken, L. E. & Amiji, M. M., Expert Opin. Drug Deliv., 3(2): 205-216 (2006)).
  • the nanoparticles also include but are not limited to viral particles and ceramic particles.
  • the attachment component can include a functional group that can be used to covalently attach the attachment component to a reactive group present on the nanoparticle.
  • the functional group can be located anywhere on the attachment component, such as, e.g., the terminal position of an alkylene or heteroalkylene moiety.
  • a wide variety of functional groups are generally known in the art and can be reacted under several classes of reactions, such as but not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides or active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction or Diels-Alder addition).
  • Suitable functional groups can include, for example: (a) carboxyl groups and various derivatives thereof including, but not limited to, N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters; (b) hydroxyl groups which can be converted to esters, ethers, aldehydes, etc.
  • haloalkyl groups wherein the halide can be later displaced with a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom;
  • dienophile groups which are capable of participating in Diels-Alder reactions such as, for example, maleimido groups;
  • aldehyde or ketone groups such that subsequent derivatization is possible via formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or via such reactions as Grignard addition or alkyllithium addition;
  • sulfonyl halide groups for subsequent reaction with amines, for example, to form sulfonamides;
  • thiol groups which can be converted to disulfides or reacted with amines, for
  • click chemistry-based platforms can be used to attach the attachment component to a nanoparticle (Kolb, H. C., M. G. Finn and K. B. Sharpless, Angew. Chem. Int'l. Ed. 40 (11): 2004 (2001)).
  • the attachment component can include one functional group or a plurality of functional groups that result in a plurality of covalent bonds with the nanoparticle.
  • Table 1 provides an additional non-limiting, representative list of functional groups that can be used in the present invention.
  • an attachment component can be attached to a nanoparticle by non-covalent interactions that can include but are not limited to affinity interactions, metal coordination, physical adsorption, hydrophobic interactions, van der Waals interactions, hydrogen bonding interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, antibody-binding interactions, hybridization interactions between complementary DNA, and the like.
  • an attachment component can be, e.g., a lipid or phospholipid, that incorporates into a nanoparticle.
  • a lipid or phospholipid can be present in a lipid bilayer portion of a nanoparticle, wherein in certain embodiments the nanoparticle is a liposome.
  • an attachment component can be a lipid or phospholipid (e.g., a C 8 -C 36 alkyl, which can be saturated or unsaturated) that interacts partially or wholly with the hydrophobic and/or hydrophilic regions of the lipid bilayer.
  • the attachment component can include one group that allows non-covalent interaction with the nanoparticle, but a plurality of groups is also contemplated. For example, a plurality of ionic charges can be used to produce sufficient non-covalent interaction between the attachment component and the nanoparticle.
  • the attachment component can include a plurality of lipids such that the plurality of lipids interacts with a bilayer membrane of a liposome or bilayer or monolayer coated on a nanoparticle.
  • surrounding solution conditions can be modified to disrupt non-covalent interactions thereby detaching the attachment component from the nanoparticle.
  • the compounds of the present invention can include R 1 , R 2 , R 3 , R 4 , R 5 and/or R 6 as an attachment component.
  • the attachment component can include a phenyl group, cholesterol, or a lipid, such as a saturated or unsaturated C 10 -C 24 alkyl group or a substituted saturated or unsaturated C 10 -C 24 alkyl group.
  • the attachment component can be selected to facilitate association of the attachment component with a lipid bilayer.
  • the length, sites and geometries of double bonds and/or substitutions of the alkyl groups can be selected to provide a desired level of incorporation with the lipid bilayer to allow modification of the surface properties of a liposome by display of other components, such as, e.g., targeting agents and/or stealth agents.
  • the phosphonate conjugates can be directly attached to a nanoparticle by way of a linking group, L 1 , L 2 , L 3 , L 4 , L 5 and/or L 6 .
  • R 1 , R 3 , R 4 , and/or R 6 can be a nanoparticle.
  • R 1 is an attachment component, and the attachment component is a phospholipid.
  • the phospholipid has the formula:
  • each of R 7 and R 8 is independently selected from a saturated or unsaturated C 10-24 alkyl or alkanoyl group and a substituted saturated or unsaturated C 10 -C 24 alkyl or alkanoyl group.
  • each of R 7 and R 8 is independently selected from saturated or unsaturated C 10 -C 18 alkyl or alkanoyl group and a substituted saturated or unsaturated C 10 -C 18 alkyl or alkanoyl group.
  • Suitable alkanoyl groups include, but are not limited to, decanoyl, undecanoyl, lauroyl, tridecanoyl, myristoyl, pentadecanoyl, palmitoyl, heptadecanoyl, stearoyl, nonadecanoyl, arachidoyl, and the like.
  • Other phospholipids generally known in the art and described herein can also be used.
  • the phosphonate conjugates can be prepared to modify the surface characteristics (e.g., hydrophilic or hydrophobic characteristics) of a nanoparticle.
  • positively and negatively charged groups can be included in the conjugates and further incorporated with a nanoparticle.
  • acidic groups can be included in the conjugate to incorporate a negative charge on the surface of a liposome.
  • the conjugates of the present invention can include a [L 2 -R 2 ] n group that can be used to modify the surface characteristics.
  • n can be an integer of 0, 1 or 2.
  • L 2 can be a bond or a linking group.
  • R 2 can include, but is not limited to, a stealth agent, C 1 -C 10 alkyl, a carboxylic acid or ester, a phosphonic acid or ester, a sulfonic acid or ester, or a hydroxy.
  • Linking groups are another feature of the phosphonate conjugates of the present invention.
  • One of ordinary skill in the art can appreciate that a variety of linking groups are known in the art and can be found, for example, in the following reference: Hermanson, G. T., Bioconjugate Techniques, 2 nd Ed., Academic Press, Inc. (2008).
  • Linking groups of the present invention can be used to provide additional properties to the compounds, such as providing spacing between different portions of the compounds. This spacing can be used, for example, to overcome steric hindrance issues caused by a nanoparticle, e.g., when a targeting agent spaced a distance away from the nanoparticle can bind to a target.
  • linking groups can be used to change the physical properties of the compounds.
  • the phosphonate conjugates of the present invention include L 1 , L 2 , L 3 , L 4 , L 5 and L 6 , which can each independently be a linking group or a bond.
  • L 1 , L 2 , L 3 , L 4 , L 5 and L 6 can each independently be selected to be a hydrophilic, non-immunogenic water soluble linking group.
  • the hydrophilic, non-immunogenic water soluble linking groups of the present invention can include, but are not limited to, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, polycarboxylate, polysaccharide, and dextran.
  • One of ordinary skill in the art will appreciate that the length and/or chemical properties of a linking group can be selected for certain applications, such as the spacing considerations discussed above.
  • the linking groups can be, for example, C 1-30 alkylene linking groups or similar heteroalkylene linking groups (an alkylene linking group in which the carbon chain is interrupted by from one to ten heteroatoms selected from O, N and S).
  • the linking groups can include an aryl moiety such as a phenylene ring or a heteroaryl counterpart.
  • linking groups can include alkylene or heteroalkylene linking groups substituted with amido, amino, keto, and hydroxyl groups.
  • the phosphonate conjugates can include at least one stealth agent.
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 can be independently selected to be a stealth agent.
  • a stealth agent can prevent nanoparticles from sticking to each other and to blood cells or vascular walls.
  • stealth nanoparticles e.g., stealth liposomes, can reduce immunogenicity and/or reactogenecity when the nanoparticles are administered to a subject.
  • Stealth agents can also increase blood circulation time of a nanoparticle within a subject.
  • a nanoparticle can include a stealth agent such that, for example, the nanoparticle is partially or fully composed of a stealth agent or the nanoparticle is coated with a stealth agent.
  • Stealth agents for use in the present invention can include those generally well known in the art. Suitable stealth agents can include but are not limited to dendrimers, polyalkylene oxide, polyethylene glycol, polyvinyl alcohol, polycarboxylate, polysaccharides, and/or hydroxyalkyl starch. Stealth agents can be attached to the phosphonate conjugates of the present invention through covalent and/or non-covalent attachment, as described above with respect to the attachment component.
  • a stealth agent can include a polyalkylene oxide, such as “polyethylene glycol,” which is well known in the art and refers generally to an oligomer or polymer of ethylene oxide.
  • Polyethylene glycol can be linear or branched, wherein branched PEG molecules can have additional PEG molecules emanating from a central core and/or multiple PEG molecules can be grafted to the polymer backbone.
  • polyethylene glycol can be produced in as a distribution of molecular weights, which can be used to identify the type of PEG. For example, PEG 500 is identified by a distribution of PEG molecules having an average molecular weight of ⁇ 500 g/mol, as measured by methods generally known in the art.
  • PEG can be represented by the following formula: H—[O—(CH 2 ) 2 ] m —OH, in which m is the number of monomers present in the polymer (e.g., m can range from 1 to 200).
  • m is the number of monomers present in the polymer (e.g., m can range from 1 to 200).
  • PEG 100 can include PEG polymers in which m is equal to 2.
  • PEG 1000 can include PEG molecules in which m is equal to 23.
  • PEG 5000 can include PEG molecules in which m is equal to 114.
  • PEG can include low or high molecular weight PEG, e.g., PEG 100 , PEG 500 , PEG 2000 , PEG 3400 , PEG 5000 , PEG 10000 , or PEG 20000 .
  • PEG can range between PEG 100 to PEG 10000 , or PEG 1000 to PEG 10000 , or PEG 1000 to PEG 5000 .
  • the stealth agent can be PEG 500 , PEG 1000 , PEG 2000 , or PEG 5000 .
  • PEG can be terminated with a methyl ether, an alcohol, or a carboxylic acid.
  • the stealth agent can include at least two PEG molecules each linked together with a linking group.
  • Linking groups can include those described above, e.g., amide linkages.
  • PEGylated-lipids are present in a bilayer of the nanoparticle, e.g., a liposome, in an amount sufficient to make the nanoparticle “stealth,” wherein a stealth nanoparticle shows reduced immunogenicity.
  • the compounds of the present invention can include a therapeutic agent, diagnostic agent, or a combination thereof.
  • the therapeutic and/or diagnostic agent can be associated directly with a phosphonate conjugate of the present invention.
  • the therapeutic and/or diagnostic agent can be covalently attached to the phosphonate conjugate.
  • the therapeutic agent and/or diagnostic agent can be present anywhere in, on, or around a nanoparticle associated with the phosphonate conjugates of the present invention.
  • the therapeutic agent and/or diagnostic agent can be embedded in, encapsulated in, or tethered to the nanoparticle.
  • the nanoparticle is a liposome and the diagnostic and/or therapeutic agent is encapsulated in the liposome.
  • a therapeutic agent used in the present invention can include any agent directed to treat a condition in a subject.
  • any therapeutic agent known in the art can be used, including without limitation agents listed in the United States Pharmacopeia (U.S.P.), Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10 th Ed., McGraw Hill, 2001; Katzung, Ed., Basic and Clinical Pharmacology , McGraw-Hill/Appleton & Lange, 8 th ed., Sep.
  • Therapeutic agents can be selected depending on the type of disease desired to be treated. For example, certain types of cancers or tumors, such as carcinoma, sarcoma, leukemia, lymphoma, myeloma, and central nervous system cancers as well as solid tumors and mixed tumors, can involve administration of the same or possibly different therapeutic agents.
  • a therapeutic agent can be delivered to treat or affect a cancerous condition in a subject and can include chemotherapeutic agents, such as alkylating agents, antimetabolites, anthracyclines, alkaloids, topoisomerase inhibitors, and other anticancer agents.
  • the agents can include antisense agents, microRNA, siRNA and/or shRNA agents.
  • a therapeutic agent can include an anticancer agent or cytotoxic agent including but not limited to avastin, doxorubicin, cisplatin, oxaliplatin, carboplatin, 5-fluorouracil, gemcitibine or taxanes, such as paclitaxel and docetaxel.
  • an anticancer agent or cytotoxic agent including but not limited to avastin, doxorubicin, cisplatin, oxaliplatin, carboplatin, 5-fluorouracil, gemcitibine or taxanes, such as paclitaxel and docetaxel.
  • Additional anti-cancer agents can include but are not limited to 20-epi-1,25 dihydroxyvitamin D3,4-ipomeanol, 5-ethynyluracil, 9-dihydrotaxol, abiraterone, acivicin, aclarubicin, acodazole hydrochloride, acronine, acylfulvene, adecypenol, adozelesin, aldesleukin, all-tk antagonists, altretamine, ambamustine, ambomycin, ametantrone acetate, amidox, amifostine, aminoglutethimide, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitors, antagonist D, antagonist G, antarelix, anthramycin, anti-dorsalizing morphogenetic protein-1, antiestrogen, antineoplaston, antis
  • the therapeutic agents can be part of a cocktail of agents that includes administering two or more therapeutic agents.
  • a liposome having both cisplatin and oxaliplatin can be administered.
  • the therapeutic agents can be delivered before, after, or with immune stimulatory adjuvants, such as aluminum gel or salt adjuvants (e.g., aluminum phosphate or aluminum hydroxide), calcium phosphate, endotoxins, toll-like receptor adjuvants and the like.
  • immune stimulatory adjuvants such as aluminum gel or salt adjuvants (e.g., aluminum phosphate or aluminum hydroxide), calcium phosphate, endotoxins, toll-like receptor adjuvants and the like.
  • Therapeutic agents of the present invention can also include radionuclides for use in therapeutic applications.
  • emitters of Auger electrons such as 111 In
  • a chelate such as diethylenetriaminepentaacetic acid (DTPA) or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)
  • DTPA diethylenetriaminepentaacetic acid
  • DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
  • radionuclide and/or radionuclide-chelate combinations can include but are not limited to beta radionuclides ( 177 Lu, 153 Sm, 88/90 Y) with DOTA, 64 Cu-TETA, 188/186 Re(CO) 3 -IDA; 188/186 Re(CO)triamines (cyclic or linear), 188/186 Re(CO) 3 -Enpy2, and 188/186 Re(CO) 3 -DTPA.
  • the therapeutic agents used in the present invention can be associated with the nanoparticle in a variety of ways, such as being embedded in, encapsulated in, or tethered to the nanoparticle. Loading of the therapeutic agents can be carried out through a variety of ways known in the art, as disclosed for example in the following references: de Villiers, M. M. et al., Eds., Nanotechnology in Drug Delivery , Springer (2009); Gregoriadis, G., Ed., Liposome Technology: Entrapment of drugs and other materials into liposomes , CRC Press (2006). In a group of embodiments, one or more therapeutic agents can be loaded into liposomes.
  • Loading of liposomes can be carried out, for example, in an active or passive manner.
  • a therapeutic agent can be included during the self-assembly process of the liposomes in a solution, such that the therapeutic agent is encapsulated within the liposome.
  • the therapeutic agent may also be embedded in the liposome bilayer or within multiple layers of multilamellar liposome.
  • the therapeutic agent can be actively loaded into liposomes.
  • the liposomes can be exposed to conditions, such as electroporation, in which the bilayer membrane is made permeable to a solution containing therapeutic agent thereby allowing for the therapeutic agent to enter into the internal volume of the liposomes.
  • a diagnostic agent used in the present invention can include any diagnostic agent known in the art, as provided, for example, in the following references: Armstrong et al., Diagnostic Imaging, 5 th Ed., Blackwell Publishing (2004); Torchilin, V. P., Ed., Targeted Delivery of Imaging Agents , CRC Press (1995); Vallabhajosula, S., Molecular Imaging: Radiopharmaceuticals for PET and SPECT , Springer (2009).
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 can be independently selected to be a diagnostic agent.
  • a diagnostic agent can be detected by a variety of ways, including as an agent providing and/or enhancing a detectable signal that includes, but is not limited to, gamma-emitting, radioactive, echogenic, optical, fluorescent, absorptive, magnetic or tomography signals.
  • Techniques for imaging the diagnostic agent can include, but are not limited to, single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, positron emission tomography (PET), computed tomography (CT), x-ray imaging, gamma ray imaging, and the like.
  • a diagnostic agent can include chelators that bind, e.g., to metal ions to be used for a variety of diagnostic imaging techniques.
  • exemplary chelators include but are not limited to ethylenediaminetetraacetic acid (EDTA), [4-(1,4,8,11-tetraazacyclotetradec-1-yl) methyl]benzoic acid (CPTA), Cyclohexanediaminetetraacetic acid (CDTA), ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), citric acid, hydroxyethyl ethylenediamine triacetic acid (HEDTA), iminodiacetic acid (IDA), triethylene tetraamine hexaacetic acid (TTHA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylene phosphonic acid) (DOTP), 1,4,8,11-tetraazacyclo
  • a radioisotope can be incorporated into some of the diagnostic agents described herein and can include radionuclides that emit gamma rays, positrons, beta and alpha particles, and X-rays.
  • Suitable radionuclides include but are not limited to 225 Ac, 72 As, 211 At, 11 B, 128 Ba, 212 Bi, 75 Br, 77 Br, 14 C, 109 Cd, 62 Cu, 64 Cu, 67 Cu, 18 F, 67 Ga, 68 Ga, 3 H, 123 I, 125 I, 130 I, 131 I, 111 In, 177 Lu, 13 N, 15 O, 32 P, 33 P, 212 Pb, 103 Pd, 186 Re, 188 Re, 47 Sc, 153 Sm, 89 Sr, 99m Tc, 88 Y and 99 Y.
  • radioactive agents can include 111 In-DTPA, 99m Tc(CO) 3 -DTPA, 99m Tc(CO) 3 -ENPy2, 62/64/67 Cu-TETA, 99m Tc(CO) 3 -IDA, and 99m Tc(CO) 3 triamines (cyclic or linear).
  • the agents can include DOTA and its various analogs with 111 In, 177 Lu, 153 Sm, 88/90 Y, 62/64/67 Cu, or 67/68 Ga.
  • the liposomes can be radiolabeled, for example, by incorporation of lipids attached to chelates, such as DTPA-lipid, as provided in the following references: Phillips et al., Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology, 1(1): 69-83 (2008); Torchilin, V. P. & Weissig, V., Eds. Liposomes 2nd Ed.: Oxford Univ. Press (2003); Elbayoumi, T. A. & Torchilin, V. P., Eur. J. Nucl. Med. Mol. Imaging 33:1196-(2006); Mougin-Degraef, M. et al., Int'l J. Pharmaceutics 344:110-117 (2007).
  • chelates such as DTPA-lipid
  • the diagnostic agents can include optical agents such as fluorescent agents, phosphorescent agents, chemiluminescent agents, and the like.
  • optical agents such as fluorescent agents, phosphorescent agents, chemiluminescent agents, and the like.
  • Numerous agents e.g., dyes, probes, labels, or indicators
  • Fluorescent agents can include a variety of organic and/or inorganic small molecules or a variety of fluorescent proteins and derivatives thereof.
  • fluorescent agents can include but are not limited to cyanines, phthalocyanines, porphyrins, indocyanines, rhodamines, phenoxazines, phenylxanthenes, phenothiazines, phenoselenazines, fluoresceins, benzoporphyrins, squaraines, dipyrrolo pyrimidones, tetracenes, quinolines, pyrazines, corrins, croconiums, acridones, phenanthridines, rhodamines, acridines, anthraquinones, chalcogenopyrylium analogues, chlorins, naphthalocyanines, methine dyes, indolenium dyes, azo compounds, azulenes, azaazulenes, triphenyl methane dyes, indoles, benzoindoles, indoc
  • agents that can be used include, but are not limited to, for example, fluorescein, fluorescein-polyaspartic acid conjugates, fluorescein-polyglutamic acid conjugates, fluorescein-polyarginine conjugates, indocyanine green, indocyanine-dodecaaspartic acid conjugates, indocyanine-polyaspartic acid conjugates, isosulfan blue, indole disulfonates, benzoindole disulfonate, bis(ethylcarboxymethyl)indocyanine, bis(pentylcarboxymethyl)indocyanine, polyhydroxyindole sulfonates, polyhydroxybenzoindole sulfonate, rigid heteroatomic indole sulfonate, indocyaninebispropanoic acid, indocyaninebishexanoic acid, 3,6-dicyano-2,5-[(N,N,N′,
  • optical agents used can depend on the wavelength used for excitation, depth underneath skin tissue, and other factors generally well known in the art.
  • optimal absorption or excitation maxima for the optical agents can vary depending on the agent employed, but in general, the optical agents of the present invention will absorb or be excited by light in the ultraviolet (UV), visible, or infrared (IR) range of the electromagnetic spectrum.
  • UV ultraviolet
  • IR infrared
  • dyes that absorb and emit in the near-IR ⁇ 700-900 nm, e.g., indocyanines
  • any dyes absorbing in the visible range are suitable.
  • the non-ionizing radiation employed in the process of the present invention can range in wavelength from about 350 nm to about 1200 nm.
  • the fluorescent agent can be excited by light having a wavelength in the blue range of the visible portion of the electromagnetic spectrum (from about 430 nm to about 500 nm) and emits at a wavelength in the green range of the visible portion of the electromagnetic spectrum (from about 520 nm to about 565 nm).
  • fluorescein dyes can be excited with light with a wavelength of about 488 nm and have an emission wavelength of about 520 nm.
  • 3,6-diaminopyrazine-2,5-dicarboxylic acid can be excited with light having a wavelength of about 470 nm and fluoresces at a wavelength of about 532 nm.
  • the excitation and emission wavelengths of the optical agent may fall in the near-infrared range of the electromagnetic spectrum.
  • indocyanine dyes such as indocyanine green, can be excited with light with a wavelength of about 780 nm and have an emission wavelength of about 830 nm.
  • the diagnostic agents can include but are not limited to magnetic resonance (MR) and x-ray contrast agents that are generally well known in the art, including, for example, iodine-based x-ray contrast agents, superparamagnetic iron oxide (SPIO), complexes of gadolinium or manganese, and the like. (See, e.g., Armstrong et al., Diagnostic Imaging, 5 th Ed., Blackwell Publishing (2004)).
  • a diagnostic agent can include a magnetic resonance (MR) imaging agent.
  • Exemplary magnetic resonance agents include but are not limited to paramagnetic agents, superparamagnetic agents, and the like.
  • Exemplary paramagnetic agents can include but are not limited to Gadopentetic acid, Gadoteric acid, Gadodiamide, Gadolinium, Gadoteridol, Mangafodipir, Gadoversetamide, Ferric ammonium citrate, Gadobenic acid, Gadobutrol, or Gadoxetic acid.
  • Superparamagnetic agents can include but are not limited to superparamagnetic iron oxide and Ferristene.
  • the diagnostic agents can include x-ray contrast agents as provided, for example, in the following references: H. S Thomsen, R. N. Muller and R. F. Mattrey, Eds., Trends in Contrast Media , (Berlin: Springer-Verlag, 1999); P.
  • x-ray contrast agents include, without limitation, iopamidol, iomeprol, iohexol, iopentol, iopromide, iosimide, ioversol, iotrolan, iotasul, iodixanol, iodecimol, ioglucamide, ioglunide, iogulamide, iosarcol, ioxilan, iopamiron, metrizamide, iobitridol and iosimenol.
  • the x-ray contrast agents can include iopamidol, iomeprol, iopromide, iohexol, iopentol, ioversol, iobitridol, iodixanol, iotrolan and iosimenol.
  • the diagnostic agents can be associated with the nanoparticle in a variety of ways, including for example being embedded in, encapsulated in, or tethered to the nanoparticle.
  • loading of the diagnostic agents can be carried out through a variety of ways known in the art, as disclosed for example in the following references: de Villiers, M. M. et al., Eds., Nanotechnology in Drug Delivery , Springer (2009); Gregoriadis, G., Ed., Liposome Technology: Entrapment of drugs and other materials into liposomes , CRC Press (2006).
  • the phosphonate conjugates of the present invention can also include at least one targeting agent.
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 can be independently selected to be a targeting agent.
  • the targeting agents of the present invention can associate with any target of interest, such as a target associated with an organ, tissues, cell, extracellular matrix, or intracellular region.
  • a target can be associated with a particular disease state, such as a cancerous condition.
  • a targeting agent can target one or more particular types of cells that can, for example, have a target that indicates a particular disease and/or particular state of a cell, tissue, and/or subject.
  • the targeting agent can be specific to only one target, such as a receptor.
  • Suitable targets can include but are not limited to a nucleic acid, such as a DNA, RNA, or modified derivatives thereof.
  • Suitable targets can also include but are not limited to a protein, such as an extracellular protein, a receptor, a cell surface receptor, a tumor-marker, a transmembrane protein, an enzyme, or an antibody.
  • Suitable targets can include a carbohydrate, such as a monosaccharide, disaccharide, or polysaccharide that can be, for example, present on the surface of a cell.
  • suitable targets can include mucins such as MUC-1 and MUC-4, growth factor receptors such as EGFR, Claudin 4, nucleolar phosphoproteins such as nucleolin, chemokine receptors such as CCR7, receptors such as somatostatin receptor 4, Erb-B2 (erythroblastic leukaemia oncogene homologue 2) receptor, CD44 receptor, and VEGF receptor-2 kinase.
  • a targeting agent can include a small molecule mimic of a target ligand (e.g., a peptide mimetic ligand), a target ligand (e.g., an RGD peptide containing peptide or folate amide), or an antibody or antibody fragment specific for a particular target.
  • a targeting agent can further include folic acid derivatives, B-12 derivatives, integrin RGD peptides, NGR derivatives, somatostatin derivatives or peptides that bind to the somatostatin receptor, e.g., octreotide and octreotate, and the like.
  • the targeting agents of the present invention can also include an aptamer.
  • Aptamers can be designed to associate with or bind to a target of interest.
  • Aptamers can be comprised of, for example, DNA, RNA, and/or peptides, and certain aspects of aptamers are well known in the art. (See. e.g., Klussman, S., Ed., The Aptamer Handbook, Wiley-VCH (2006); Nissenbaum, E. T., Trends in Biotech. 26(8): 442-449 (2008)).
  • suitable aptamers can be linear or cyclized and can include oligonucleotides having less than about 150 bases (i.e., less than about 150 mer).
  • Aptamers can range in length from about 100 to about 150 bases or from about 80 to about 120 bases. In certain embodiments, the aptamers can range from about 12 to 40 about bases, from about 12 to about 25 bases, from about 18 to about 30 bases, or from about 15 to about 50 bases.
  • the aptamers can be developed for use with a suitable target that is present or is expressed at the disease state, and includes, but is not limited to, the target sites noted herein.
  • the present invention includes a method of preparing a phosphonate conjugate, the method comprising: combining a primary amine compound having the formula: H 2 N(L 1 )-(R 1 ), a carbonyl compound having the formula: O ⁇ C[(L 2 )-(R 2 )] n , and a H-phosphonate compound having the formula:
  • each L 1 , L 2 , L 3 and L 4 is independently selected from the group consisting of a bond and a linking group;
  • R 1 is selected from the group consisting of a nanoparticle, an attachment component, a targeting agent, a diagnostic agent, a stealth agent, and a tetrapodal presentation component;
  • each R 2 is independently selected from the group consisting of a stealth agent, C 1 -C 10 alkyl, a carboxylic acid or ester, a phosphonic acid or ester, a sulfonic acid or ester, and a hydroxy;
  • each R 3 and R 4 is independently selected from the group consisting of H, an attachment component, a targeting agent, a diagnostic agent, and a stealth agent, wherein at least one of R 3 or R 4 is other than H; and
  • n is an integer from 0 to 2, wherein when n is 0 or 1 the carbonyl compound is an aldehyde.
  • the present invention includes a method of preparing a phosphonate conjugate.
  • the method includes combining a secondary amine compound having the formula: HN[(L 1 )-(R 1 )](R 9 ), a carbonyl compound having the formula: O ⁇ C[(L 2 )-(R 2 )] n , and a H-phosphonate compound having the formula:
  • each L 1 , L 2 , L 3 and L 4 is independently selected from the group consisting of a bond and a linking group;
  • R 1 is selected from the group consisting of a nanoparticle, an attachment component, a targeting agent, a diagnostic agent and a stealth agent;
  • R 2 is independently selected from the group consisting of a stealth agent, C 1 -C 10 alkyl, a carboxylic acid or ester, a phosphonic acid or ester, a sulfonic acid or ester, and a hydroxy;
  • each R 3 and R 4 is independently selected from the group consisting of H, an attachment component, a targeting agent, a diagnostic agent, and a stealth agent, wherein at least one of R 3 or R 4 is other than H;
  • the reactions used to make the phosphonate conjugates include a H-phosphonate compound, a carbonyl compound, and a primary amine compound or a secondary amine compound. These compounds can be combined in any order during synthesis to reach the final phosphonate conjugates.
  • the primary amine compound or secondary amine compound and the carbonyl compound can be combined to form an intermediate compound before being combined with a H-phosphonate compound to form the final phosphonate conjugate.
  • a H-phosphonate compound can be combined with a carbonyl compound to form an intermediate compound before being combined with a primary amine compound or secondary amine compound to form the final phosphonate conjugate.
  • the necessary compounds can be combined together in one reaction to produce the final phosphonate conjugates.
  • the ratio of a H-phosphonate compound, a carbonyl compound, and a primary amine compound can be 2:2:1.
  • the ratio of a H-phosphonate compound, a carbonyl compound, and a secondary amine compound can be 1:1:1. The ratio of the different compounds will depend on the particular reaction conditions and desired phosphonate conjugates.
  • the reactions involving a H-phosphonate compound, a primary or a secondary amine compound, and a carbonyl compound can be used to produce a large variety of compounds that can include an attachment component, a targeting agent, a diagnostic agent, a therapeutic agent, a stealth agent, or a combination thereof.
  • a H-phosphonate compound, a carbonyl compound, and a primary amine compound at ratios of 2:2:1 can be combined to form phosphonate conjugates of the present invention.
  • the phosphonate conjugate can include R 1 as a PEG group ranging from PEG 1000 -PEG 10000 .
  • R 3 and R 4 can also be selected from C 1 -C 20 alkyl groups to facilitate, for example, attachment to a bilayer membrane of a liposome.
  • n is zero in which the carbonyl compound then is formaldehyde.
  • the carbonyl compound can have n as 1 and R 2 as a methyl group, which changes the structure of the phosphonate conjugate in FIG. 1 .
  • One of ordinary skill in the art will appreciate the variety of possible combinations that can be used to produce the scope of phosphonate conjugates disclosed herein.
  • phosphonate conjugates can be produced so as to provide a presentation assembly that can extend, e.g., from the surface of a nanoparticle.
  • a lipid molecule can include a primary amine and R 7 and R 8 , which can be, e.g., C 1 -C 20 alkyl groups.
  • R 7 and R 8 can be, e.g., C 1 -C 20 alkyl groups.
  • n is 0 and L 3 and L 4 can be a bond or a short alkylene group (e.g., C 1 -C 6 ) or other linking group to allow for desired spacing or other characteristics desired for a particular application.
  • the lipid molecule can be combined with a H-phosphonate compound and formaldehyde at a ratio of 1:2:2, respectively, to form a phosphonate conjugate presenting a tetrapodal assembly of R 3 and R 4 .
  • the methods of making these compounds can be combined with nanoparticles to allow synthesis of the phosphonate conjugates while one of the starting materials, e.g., a H-phosphonate compound, is attached to the nanoparticle.
  • a H-phosphonate compound e.g., a H-phosphonate compound
  • two adjacent H-phosphonate compounds in a lipid bilayer membrane can be combined with two equivalents of a carbonyl compound, O ⁇ C[(L 2 )-(R 2 )] n , and one equivalent of a primary amine compound, H 2 N(L 1 )-(R 1 ), to produce the phosphonate conjugates of the present invention on the surface of a nanoparticle.
  • a carbonyl compound O ⁇ C[(L 2 )-(R 2 )] n
  • H 2 N(L 1 )-(R 1 ) a primary amine compound
  • R 4 , R 3 and R 4 can be attachment components, e.g., a C 1 -C 20 alkyl group, that is embedded in the lipid bilayer of a liposome.
  • the prepared liposomes having the H-phosphonate compound can then be combined with the primary amine compound, which, e.g., includes a stealth agent, such as PEG ranging from PEG 100 -PEG 10000 . After a hydrophosphonylation reaction including the carbonyl compound, the stealth agent can be displayed on the surface of the liposome, thereby transforming the liposome to a stealth liposome.
  • a lipid molecule can include a primary amine that extends from the surface, and R 7 and R 8 can be, e.g., C 1 -C 20 alkyl groups that embed in the lipid bilayer.
  • R 7 and R 8 can be, e.g., C 1 -C 20 alkyl groups that embed in the lipid bilayer.
  • a hydrophosphonylation reaction can form a bridge between the two H-phosphonate compounds and also present R 5 and R 6 on the surface of the liposome, in which R 5 and R 6 can be a stealth agent, such as PEG ranging from PEG 100 -PEG 10000 .
  • linking groups L 1 , L 2 , and L 3 can be independently selected as a linking group or a bond to allow for desired spacing or other characteristics desired for a particular application.
  • One of ordinary skill in the art will appreciate the variety of synthetic methods that can be used to produce the phosphonate conjugates of the present invention on the surface of a nanoparticle.
  • the starting materials and the intermediates of the synthetic reaction schemes described herein can be isolated and purified if desired using conventional techniques, including but not limited to, filtration, distillation, crystallization, chromatography, and the like. Such materials can be characterized using conventional means, including physical constants and spectral data.
  • the reactions described herein can be conducted under an inert atmosphere at atmospheric pressure at a reaction temperature range of from about ⁇ 78° C. to about 150° C., or from about 0° C. to about 125° C., and in some embodiments at about room (or ambient) temperature, e.g., about 20° C.
  • reaction solvents can include an organic solvents, such as an aprotic solvent (e.g., THF or ether).
  • reaction solvents can be aqueous solvents and can include additional buffers, salts, additives, and the like.
  • Certain reactions described herein can include reflux conditions to, for example, purify certain compounds described herein.
  • the hydrophosphonylation reactions provide several advantages for producing the phosphonate conjugates of the present invention on a nanoparticle
  • other methods can be used to make the compounds.
  • the H-phosphonate compounds, primary and secondary amine compounds, and carbonyl compounds can be reacted together to form the phosphonate conjugates of the present invention.
  • the phosphonate conjugates can be attached to a nanoparticle.
  • the phosphonate conjugates can be incorporated into liposomes by first producing the liposomes using standard methods, e.g., extrusion, and subsequently attaching the phosphonate conjugates to the liposomes.
  • the phosphonate conjugates can be incorporated into the liposome bilayer during formation of the liposomes by, e.g., drying the phosphonate conjugates and lipid components together and then resuspending the mixture in aqueous solution to form the liposomes with the phosphonate conjugates associated with the bilayer.
  • the phosphonate conjugates can be produced using other synthetic sequences.
  • a primary amine compound including R 1 and L 1 as shown in FIG. 1 , can be reacted with a H-phosphonate that does not contain R 3 and R 4 .
  • L 3 and L 4 can be linking groups that include a functional group for bonding to an R 3 and/or R 4 , which can include, e.g., a targeting agent, a stealth agent, or a diagnostic agent.
  • R 3 and/or R 4 can be reacted with the functional group of L 3 and L 4 to produce a final phosphonate conjugate.
  • R 3 and R 4 can be the same and thus, for example, if R 3 and R 4 are each a targeting agent, then L 3 and L 4 can each contain functional groups that can react with the targeting agent to produce a phosphonate conjugate of the present invention.
  • the present invention includes the use of nanoparticles that can be produced by a variety of ways generally known in the art and methods of making such nanoparticles can depend on the particular nanoparticle desired. Any measuring technique available in the art can be used to determine properties of the targeted delivery compositions and nanoparticles. For example, techniques such as dynamic light scattering, x-ray photoelectron microscopy, powder x-ray diffraction, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) can be used to determine average size and dispersity of the nanoparticles and/or targeted delivery compositions.
  • SEM scanning electron microscopy
  • TEM transmission electron microscopy
  • AFM atomic force microscopy
  • Liposomes used in the present invention can be made using a variety of techniques generally well known in the art. (See, e.g., Williams, A. P., Liposomes: A Practical Approach, 2 nd Edition, Oxford Univ. Press (2003); Lasic, D. D., Liposomes in Gene Delivery, CRC Press LLC (1997)).
  • liposomes can be produced by but are not limited to techniques such as extrusion, agitation, sonication, reverse phase evaporation, self-assembly in aqueous solution, electrode-based formation techniques, microfluidic directed formation techniques, and the like.
  • methods can be used to produce liposomes that are multilamellar and/or unilamellar, which can include large unilamellar vesicles (LUV) and/or small unilamellar vesicles (SUV).
  • micelles can be produced using techniques generally well known in the art, such that amphiphilic molecules will form micelles when dissolved in solution conditions sufficient to form micelles.
  • Lipid-coated bubbles and lipoproteins can also be constructed using methods known in the art (See, e.g., Farook, U., J. R. Soc. Interface, 6(32): 271-277 (2009); Lacko et al., Lipoprotein Nanoparticles as Delivery Vehicles for Anti - Cancer Agents in Nanotechnology for Cancer Therapy, CRC Press (2007)).
  • polymeric nanoparticles that can be used in the present invention are generally well known in the art (See, e.g., Sigmund, W. et al., Eds., Particulate Systems in Nano- and Biotechnologies, CRC Press LLC (2009); Karnik et al., Nano Lett., 8(9): 2906-2912 (2008)).
  • block copolymers can be made using synthetic methods known in the art such that the block copolymers can self-assemble in a solution to form polymersomes and/or block copolymer micelles.
  • Niosomes are known in the art and can be made using a variety of techniques and compositions (Baillie A. J. et al., J. Pharm.
  • Magnetic and/or metallic particles can be constructed using any method known in the art, such as co-precipitation, thermal decomposition, and microemulsion. (See also Nagarajan, R. & Hatton, T. A., Eds., Nanoparticles Synthesis, Stabilization, Passivation, and Functionalization, Oxford Univ. Press (2008)). Gold particles and their derivatives can be made using a variety of techniques generally known in the art, such as the Turkevich method, House method, Perraut Method or sonolysis (See also, Grzelczak et al., Chem. Soc. Rev., 37: 1783-1791 (2008)).
  • the attachment component can be attached through sulfur-gold tethering chemistry.
  • Quantum dots or semiconductor nanocrystals can be synthesized using any method known in the art, such as colloidal synthesis techniques. Generally, quantum dots can be composed of a variety of materials, such as semiconductor materials including cadmium selenide, cadmium sulfide, indium arsenide, indium phosphide, and the like.
  • the phosphonate conjugates of the present invention can include components, such as targeting agents, stealth agents, diagnostic agents, therapeutic agents, and attachment components.
  • components such as targeting agents, stealth agents, diagnostic agents, therapeutic agents, and attachment components.
  • targeting agents, stealth agents, diagnostic agents, therapeutic agents can be attached to the phosphonate conjugates of the present invention through covalent and/or non-covalent attachment, as described above with respect to the attachment component.
  • the targeting agent can include an aptamer.
  • Aptamers for a particular target can be indentified using techniques known in the art, such as but not limited to, in vitro selection processes, such as SELEXTM (systematic evolution of ligands by exponential enrichment), or MonoLexTM technology (single round aptamer isolation procedure for AptaRes AG), in vivo selection processes, or combinations thereof.
  • in vitro selection processes such as SELEXTM (systematic evolution of ligands by exponential enrichment), or MonoLexTM technology (single round aptamer isolation procedure for AptaRes AG
  • in vivo selection processes or combinations thereof.
  • the above mentioned methods can be used to identify particular DNA or RNA sequences that can be used to bind a particular target site of interest, as disclosed herein.
  • the aptamer can be constructed in a variety of ways known in the art, such as phosphoramidite synthesis.
  • phosphoramidite synthesis For peptide aptamers, a variety of identification and manufacturing techniques can be used (See e.g., Colas, P., J. Biol. 7:2 (2008); Woodman, R. et al., J. Mol. Biol. 352(5): 1118-33 (2005).
  • Aptamers can be attached to the H-phosphonate compounds, primary and secondary amine compounds, and the carbonyl compounds by a variety of ways.
  • linking groups on the H-phosphonate compounds and/or primary or secondary amine compounds can be reacted with a 3′ or 5′ end of the aptamer.
  • the aptamer can be synthesized sequentially by adding one nucleic acid at a time to a linking group on the H-phosphonate compounds and primary or secondary amine compounds.
  • One of ordinary skill in the art will appreciate the well known techniques that can be used to include aptamers in the phosphonate conjugates of the present invention.
  • the present invention also includes targeted delivery compositions that include a phosphonate conjugate.
  • the present invention includes a targeted delivery composition comprising a phosphonate conjugate described herein, wherein each of R 3 and R 4 is an attachment component attached to a nanoparticle, and R 1 is selected from a targeting agent, a diagnostic agent and a stealth agent.
  • the attachment component can attach to a nanoparticle in several ways, for example, the attachment component can be a lipid that associates with a bilayer of a liposome.
  • the targeted delivery composition can include a liposome and each of R 3 and R 4 can be associated with a bilayer of the liposome and can independently be a lipid or cholesterol.
  • R 1 of the phosphonate conjugates can be a nanoparticle or an attachment component attached to a nanoparticle and each of R 3 and R 4 can be a targeting agent, a diagnostic agent or a stealth agent.
  • the targeted delivery compositions and methods of the present invention can be used for treating and/or diagnosing any disease, disorder, and/or condition associated with a subject.
  • the methods of the present invention include a method for treating or diagnosing a cancerous condition in a subject, comprising administering to the subject a targeted delivery composition including a phosphonate conjugate of the present invention attached to a nanoparticle, wherein the composition also includes a therapeutic or diagnostic agent that is sufficient to treat or diagnose the condition and at least one of R 1 , R 3 and R 4 is a targeting agent.
  • the cancerous condition can include cancers that sufficiently express (e.g., on the cell surface or in the vasculature) a receptor that is being targeted by a targeting agent of a targeted delivery composition of the present invention.
  • the methods of the present invention include a method of determining the suitability of a subject for a targeted therapeutic treatment, comprising administering to the subject a targeted delivery composition that includes a nanoparticle and a phosphonate conjugate described herein, wherein the phosphonate conjugate or nanoparticle comprises a diagnostic agent, and imaging the subject to detect the diagnostic agent, and at least one of R 1 , R 3 and R 4 is a targeting agent.
  • the present invention can include a targeted delivery composition and a physiologically (i.e., pharmaceutically) acceptable carrier.
  • a physiologically (i.e., pharmaceutically) acceptable carrier refers to a typically inert substance used as a diluent or vehicle for a drug such as a therapeutic agent. The term also encompasses a typically inert substance that imparts cohesive qualities to the composition. Typically, the physiologically acceptable carriers are present in liquid form.
  • liquid carriers examples include physiological saline, phosphate buffer, normal buffered saline (135-150 mM NaCl), water, buffered water, 0.4% saline, 0.3% glycine, glycoproteins to provide enhanced stability (e.g., albumin, lipoprotein, globulin, etc.), and the like. Since physiologically acceptable carriers are determined in part by the particular composition being administered as well as by the particular method used to administer the composition, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (See, e.g., Remington: The Science and Practice of Pharmacy, 21st edition, 2005).
  • compositions of the present invention may be sterilized by conventional, well-known sterilization techniques or may be produced under sterile conditions.
  • Aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
  • the compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.
  • Sugars can also be included for stabilizing the compositions, such as a stabilizer for lyophilized targeted delivery compositions.
  • the targeted delivery composition of choice can be made into aerosol formulations (i.e., they can be “nebulized”) to be administered via inhalation.
  • Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • Suitable formulations for rectal administration include, for example, suppositories, which includes an effective amount of a packaged targeted delivery composition with a suppository base.
  • Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons.
  • gelatin rectal capsules which contain a combination of the targeted delivery composition of choice with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Injection solutions and suspensions can also be prepared from sterile powders, granules, and tablets.
  • compositions can be administered, for example, by intravenous infusion, topically, intraperitoneally, intravesically, or intrathecally.
  • Parenteral administration and intravenous administration are the preferred methods of administration.
  • the formulations of targeted delivery compositions can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials.
  • the pharmaceutical preparation is preferably in unit dosage form.
  • the preparation is subdivided into unit doses containing appropriate quantities of the active component, e.g., a targeted delivery composition.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation.
  • the composition can, if desired, also contain other compatible therapeutic agents.
  • the targeted delivery compositions including a therapeutic and/or diagnostic agent utilized in the pharmaceutical compositions of the present invention can be administered at the initial dosage of about 0.001 mg/kg to about 1000 mg/kg daily.
  • the dosages may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the targeted delivery composition being employed. For example, dosages can be empirically determined considering the type and stage of cancer diagnosed in a particular patient.
  • the dose administered to a patient should be sufficient to affect a beneficial therapeutic response in the patient over time.
  • the size of the dose will also be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular targeted delivery composition in a particular patient. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the targeted delivery composition. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired.
  • the targeted delivery compositions of the present invention may be used to diagnose a disease, disorder, and/or condition.
  • the targeted delivery compositions can be used to diagnose a cancerous condition in a subject, such as lung cancer, breast cancer, pancreatic cancer, prostate cancer, cervical cancer, ovarian cancer, colon cancer, liver cancer, esophageal cancer, and the like.
  • methods of diagnosing a disease state may involve the use of the targeted delivery compositions to physically detect and/or locate a tumor within the body of a subject.
  • tumors can be related to cancers that sufficiently express (e.g., on the cell surface or in the vasculature) a receptor that is being targeted by a targeting agent of a targeted delivery composition of the present invention.
  • the targeted delivery compositions can also be used to diagnose diseases other than cancer, such as proliferative diseases, cardiovascular diseases, gastrointestinal diseases, genitourinary disease, neurological diseases, musculoskeletal diseases, hematological diseases, inflammatory diseases, autoimmune diseases, rheumatoid arthritis and the like.
  • the targeted delivery compositions of the invention can include a diagnostic agent that has intrinsically detectable properties.
  • the targeted delivery compositions, or a population of particles with a portion being targeted delivery compositions can be administered to a subject.
  • the subject can then be imaged using a technique for imaging the diagnostic agent, such as single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, positron emission tomography (PET), computed tomography (CT), x-ray imaging, gamma ray imaging, and the like.
  • SPECT single photon emission computed tomography
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • CT computed tomography
  • x-ray imaging gamma ray imaging
  • gamma ray imaging gamma ray imaging
  • the incorporation of a radioisotope for imaging in a particle allows in vivo tracking of the targeted delivery compositions in a subject.
  • the biodistribution and/or elimination of the targeted delivery compositions can be measured and optionally be used to alter the treatment of patient.
  • more or less of the targeted delivery compositions may be needed to optimize treatment and/or diagnosis of the patient.
  • the targeted delivery compositions of the present invention can be delivered to a subject to release a therapeutic or diagnostic agent in a targeted manner.
  • a targeted delivery composition can be delivered to a target in a subject and then a therapeutic agent embedded in, encapsulated in, or tethered to the targeted delivery composition, such as to the nanoparticle, can be delivered based on solution conditions in vicinity of the target. Solution conditions, such as pH, salt concentration, and the like, may trigger release over a short or long period of time of the therapeutic agent to the area in the vicinity of the target.
  • an enzyme can cleave the therapeutic or diagnostic agent from the targeted delivery composition to initiate release.
  • the targeted delivery compositions can be delivered to the internal regions of a cell by endocytosis and possibly later degraded in an internal compartment of the cell, such as a lysosome.
  • targeted delivery of a therapeutic or diagnostic agent can be carried out using a variety of methods generally known in the art.
  • kits for administering the targeted delivery compositions to a subject for treating and/or diagnosing a disease state typically include two or more components necessary for treating and/or diagnosing the disease state, such as a cancerous condition.
  • Components can include targeted delivery compositions of the present invention, reagents, containers and/or equipment.
  • a container within a kit may contain a targeted delivery composition including a radiopharmaceutical that is radiolabeled before use.
  • the kits can further include any of the reaction components or buffers necessary for administering the targeted delivery compositions.
  • the targeted delivery compositions can be in lyophilized form and then reconstituted prior to administration.
  • kits of the present invention can include packaging assemblies that can include one or more components used for treating and/or diagnosing the disease state of a patient.
  • a packaging assembly may include a container that houses at least one of the targeted delivery compositions as described herein.
  • a separate container may include other excipients or agents that can be mixed with the targeted delivery compositions prior to administration to a patient.
  • a physician may select and match certain components and/or packaging assemblies depending on the treatment or diagnosis needed for a particular patient.
  • the starting materials and reagents used in preparing these compounds generally are either available from commercial suppliers, such as Aldrich Chemical Co., or are prepared by methods generally known to those skilled in the art following procedures set forth in the scientific and patent literature and references, such as Fieser and Fieser's Reagents for Organic Synthesis ; Wiley & Sons: New York, 1991, Volumes 1-15; Rodd's Chemistry of Carbon Compounds , Elsevier Science Publishers, 1989, Volumes 1-5 and Supplementals; and Organic Reactions , Wiley & Sons: New York, 1991, Volumes 1-40.
  • the following synthetic reaction schemes are merely illustrative of some methods by which the compounds of the present invention can be synthesized, and various modifications to these synthetic reaction schemes can be made and will be suggested to one skilled in the art having referred to the disclosure contained in this Application.
  • the starting materials and the intermediates of the synthetic reaction schemes can be isolated and purified if desired using conventional techniques, including but not limited to, filtration, distillation, crystallization, chromatography, and the like. Such materials can be characterized using conventional means, including physical constants and spectral data.
  • dialkyl hydrogen phosphonates A generalized synthetic procedure was used to produce dialkyl hydrogen phosphonates that could be further reacted to form some of the compounds of the present invention. As depicted in FIG. 6 , the general reaction scheme was to combine diphenyl phosphite with a 1-alkanol to form the dialkyl hydrogen phosphonates.
  • the following dialkyl hydrogen phosphonates were prepared as follows:
  • reaction mixture was transferred into 100 mL round bottom flask and rotary evaporated at 70° C. to remove solvents.
  • the crude product residue was purified by normal phase silica gel flash chromatography using hexanes/ethyl acetate gradient (C 12 product) or precipitation in acetone (C 14 through C 22 products). In each case, MS and NMR data was consistent with desired product.
  • the reaction showed ca. 94% (by integration) product, 1.5% intermediate, and 4.6% of a peak at 5.5 ppm.
  • the mixture was cooled to 40-50° C., and ca. 50 mL dichloromethane was slowly added to prevent the material from solidifying and seizing the stirrer.
  • the mixture was further diluted with 150 mL dichloromethane and transferred to a separatory funnel.
  • the mixture was washed with 50 mL water, extracted with 3 ⁇ 75 mL 3N hydrochloric acid, and further washed with 75 mL each of water and brine.
  • the organic layer was dried over anhydrous Na 2 SO 4 and concentrated on the rotary evaporator (rotovap) to give a clear liquid that slowly solidifies upon standing.
  • the crude reaction mixture in a 250 mL distillation flask was placed in a Kugelrohr distillation apparatus and the volatiles removed by placing the flask under high vacuum and gradually warming the Kugelrohr oven in 10° C. increments from 60° C. to 120° C. over approximately a 1.5 hour period and holding at 120° C. for 3 hours.
  • the distillate receiver was cooled in a dry ice/acetone bath. After completing the distillation, the hot, liquefied product in the 250 mL distillation flask was poured into a tarred 125 mL glass storage jar, in which it solidified upon cooling. This afforded the product as a white waxy solid.
  • the mixture was further diluted with 150 mL of chloroform and transferred to a separatory funnel.
  • the mixture was washed with 50 mL water, extracted with 3 ⁇ 50 mL 3N hydrochloric acid, and further washed with 50 mL each of water and brine.
  • the organic layer was dried over anhydrous Na 2 SO 4 and concentrated on the rotovap to give a viscous orange oil (this slowly solidifies upon standing).
  • FIG. 7 shows the general reaction scheme for preparing Tetrapodal C 18 -PEG 1000 -OMe, which was prepared by first making dioctadecyl hydrogen phosphonate as described in Example 2 above. Next, the Tetrapodal C 18 -PEG 1000 -OMe was produced as follows: A 10 mL Kontes microflex vial equipped with a triangular stirring vane was charged with dioctadecyl hydrogen phosphonate (1.0 g, 1.704 mmol, 2 eq.), paraformaldehyde (76.7 mg, 2.56 mmol, 3 eq.), and monodispersed PEG 1000 (Quanta Biodesign, 0.927 g, 0.852 mmol, 1 eq.).
  • the cap was screwed on tightly and the vial was warmed to 75° C. and stirred for 24 to 72 hours. Periodically, the reaction was sampled for analysis using reverse phase (C4, 300 A) HPLC equipped with an evaporative light scattering detector (ELSD). If the reaction was deemed incomplete, additional paraformaldehyde may be added to aid in further conversion to the desired product. If the reaction has reached sufficient conversion to product, the reaction was cooled down and the resulting waxy solid was carried into purification.
  • reverse phase C4, 300 A
  • ELSD evaporative light scattering detector
  • the crude reaction material was dissolved in isopropyl alcohol using sonication and slight heating.
  • the crude solution typically 10-15 mg/mL was purified by reverse phase chromatography on a Waters column (C4, 300 A, 5 ⁇ m, 19 ⁇ 150 mm) with a flow rate of 30 mL/min.
  • the solvents used were acetonitrile (0.05% TFA) and water (0.05% TFA). Below is the gradient method used;
  • Tetrapodal C 18 -PEG 2000 -OMe The synthetic steps for producing Tetrapodal C 18 -PEG 2000 -OMe were similar to those disclosed above in Example 5.
  • dioctadecyl hydrogen phosphonate was prepared via the same method disclosed in Example 2.
  • Tetrapodal C 18 -PEG 2000 -OMe was produced as follows: A 10 mL Kontes microflex vial equipped with a triangular stirring vane was charged with dioctadecyl hydrogen phosphonate (0.587 g, 1.0 mmol, 2 eq.), paraformaldehyde (90.1 mg, 3.0 mmol, 6 eq.), and polydispersed PEG 2000 (NANOCS) (1.0 g, 0.50 mmol, 1 eq.).
  • the cap was screwed on tightly and the vial was warmed to 75° C. and stirred for 24 to 72 hours. Periodically a small sample was withdrawn, dissolved in isopropanol (IPA)/water and analyzed using reverse phase (C4, 300 A) high performance liquid chromatograph (HPLC) equipped with an ELSD detector. If the reaction was deemed incomplete, additional paraformaldehyde may be added to aid in further conversion to the desired product. If the reaction has reached sufficient conversion to product, the reaction was cooled down and the resulting waxy solid was carried into purification.
  • IPA isopropanol
  • HPLC high performance liquid chromatograph
  • the crude reaction material was dissolved in IPA/water ( ⁇ 3-5% water) using sonication and slight heating.
  • the crude solution was typically 15-25 mg/mL.
  • Desired product was obtained by reverse phase chromatography on a column containing C4 silica packing (5 ⁇ m, 300 A).
  • the solvents used were ACN (0.05% trifluoroacetic acid (TFA)) and water (0.05% TFA).
  • TFA trifluoroacetic acid
  • Tetrakis(tetradecyl) (((2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxatriheptacontan-73-yl)azanediyl)bis(methylene))bis(phosphonate) (Referred to Herein as Tetrapodal C 14 -PEG 1000 -OMe)
  • tetrapodal alkyl-PEG phosphonate conjugates can also be prepared.
  • dialkyl (e.g., C 12 , C 16 , and C 22 ) hydrogen phosphonates can be prepared.
  • C 12 -, C 16 -, and C 22 -PEG 1000 -OMe phosphonate conjugates can then prepared using substantially the procedure described in Example 6.
  • the reaction can include 1-dodecanol, 1-hexadecanol and 1-docosanol, respectively, for C 12 -, C 16 -, and C 22 -PEG 1000 -OMe conjugates, each of which is shown below.
  • HRMS and/or LRMS and 31 P NMR were consistent with the desired structure of each of these derivatives.
  • conjugates having varying lengths of PEG molecules can be prepared following some of the steps described above.
  • PEG 100 to PEG 10000 or higher MW PEGs can be prepared following some of the steps described above.
  • Tetrapodal C 18 phosphono-methyl amino-PEG 5000 -OMe conjugates can be prepared following the steps in Example 6, except that polydispersed PEG 5000 is used instead of PEG 1000 .
  • the chemical structure for the Tetrapodal C 18 phosphono-methyl amino-PEG 5000 -OMe conjugates is as follows:
  • DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 0.45 g, 0.60 mmol), di(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yl) phosphonate (1.34 g, 1.24 mmol) prepared in Example 9, paraformaldehyde (0.054 g, 1.81 mmol of formaldehyde), and dimethylformamide (2.0 mL) were added to a scintillation vial. The vial was capped and heated at 75° C. for 24 hours. The reaction mixture was stripped, dissolved in methanol and treated with Dowex WBA resin (1.0 g).
  • This compound was prepared using the following procedure. 2-methoxyethylamine (3 g, 40 mmol; 3.44 mL) was combined with formaldehyde (2.4 g; 320 mmol, 6.5 mL of 37% in H 2 O) and dimethylformamide (DMF) (30 mL). Diphenylphosphite (DPP) (20.6 g, 88 mmol; 2.2 equiv.) was added to the mixture slowly. After a short initial exothermic reaction, the mixture was stirred for 12 hours at room temperature. HPLC indicated complete reaction and then DMF was evaporated. The residue was dissolved in ethyl acetate (EA) (200 mL) and washed with sat.
  • EA ethyl acetate
  • This molecule was prepared using substantially the procedure of Example 11, but ditetradecyl hydrogen phosphonate was employed in place of diphenylphosphite (DPP) and the reaction was run without DMF solvent (i.e. solvent free) as in Example 5 using a reaction vial (10 mL) and teflon coated spin vane with a work-up and isolation as described in Example 5.
  • DPP diphenylphosphite
  • This compound was prepared as follows: Dioctadecyl phosphonate (5.87 g, 10 mmol) was reacted with ⁇ -alanine (0.45 g, 5 mmol) and formaldehyde (6 mL of 37% solution in water) in DMF (20 mL) at 80° C. with stirring for 2 days in a pressure bottle. The reaction mixture was removed and volatiles were evaporated carefully and the residue was dissolved in CHCL 3 (200 mL). The organic layer was washed with 10% KHSO 4 and brine (50 ml each). The organic phase was dried over MgSO 4 , filtered and evaporated to an oil.
  • This compound was prepared by reacting dioctadecyl phosphonate with glycine and formaldehyde in DMF, similar to Example 13. The product was isolated in a similar manner as shown in Example 13. The structure was confirmed by 31 P NMR and HRMS.
  • Tetraoctadecyl (78-methyl-75-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-74,78-diazanonaheptacontan-79-yl)bis(methylene)diphosphonate.
  • Dioctadecyl (78-methyl-75-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-74,78-diazanonaheptacontan-79-yl) phosphonate (C 18 /N-methyl beta-alanine/PEG 1000 ).
  • This compound can be synthesized by reacting 3-(bis((bis(octadecyloxy)phosphoryl)methyl)amino)propanoic acid formed in Example 13 with m-dPEG 24 -amine in the presence of TBTU using substantially the same reaction conditions and purification procedures outlined in Example 15.
  • Dioctadecyl (78-carboxyl-75-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-74,78-diazanonaheptacontan-79-yl) phosphonate (C 18 /N-methyl beta-alanine/PEG 1000 carboxylate) Chemical Formula: C 92 H 185 N 2 O 30 P, Exact Mass: 1829.27, Molecular Weight: 1830.42 g/mol
  • This compound may be synthesized by reacting 3-(bis((bis(octadecyloxy)phosphoryl)methyl)amino)propanoic acid formed in Example 13 with amino-PEG 24 -caboxylate ester in the presence of TBTU using substantially the same reactions conditions and purification procedures outlined in Example 15 to obtain the ester. Hydrolysis of the ester to the acid may be achieved under basic or acidic conditions and the product carboxylate isolated by C4 reverse phase HPLC, combining like fractions and lyophilizing to obtain substantially pure product.
  • This compound may be prepared by taking the product of Example 15 and reacting it with amino-PEG OMe (1000 MW) using substantially the coupling procedure and purification scheme of Example 15.
  • This compound was prepared as follows: Dicholesteryl hydrogen phosphonate (5.0 g, 0.006 mol) was reacted with sarcosine (0.54 g, 0.006 mol) and formaldehyde (2.2 g, 0.03 mol) in DMF at 75° C. as a heterogeneous mixture. After 4 hours at 75° C., a 9% conversion to desired dicholesterolphosphonyl methyl sarcosine was obtained. Another portion of formaldehyde (2.2 g, 0.03 mol) was added and the reaction temperature increased to 85° C.
  • This compound was prepared as follows: To a RBF (250 mL) equipped with mechanical stirrer and thermocouple were added dioctadecyl hydrogen phosphonate (1.98 g 0.003 mol), sarcosine (0.30 g, 0.003 mol) and of formaldehyde (1.1 g, 0.013 mol) in N,N-dimethylformamide (50 mL) at 75° C. After 2 hours, a 76% conversion to 2-((bis(octadecyloxy)phosphoryl)(methyl)amino)acetic acid was observed by 31 P NMR (24.4 ppm). The reaction was allowed to continue for another 4 hours at 75° C. producing an 88.5% conversion.
  • This compound was prepared using substantially the same procedure as in Example 20 but methyl beta-alanine was substituted for sarcosine. After C4 reverse HPLC purification and isolation, the expected MS and 31 P NMR was obtained.
  • This compound was prepared as follows: To a RBF (250 mL) equipped with mechanical stirrer and thermocouple were added dioleyl hydrogen phosphonate (1.96 g, 0.003 mol), sarcosine (0.30 g, 0.003 mol) and formaldehyde (1.1 g, 0.013 mol) in N,N-dimethylformamide (50 mL) at 75° C. The reaction was allowed to take place for 4 hours at 75° C. An approximate 89% conversion to desired 2-((bis((Z)-octadec-9-enyloxy)phosphoryl)(methyl)amino)acetic acid was observed with 31 P NMR ⁇ 24 ppm. Allowed reaction mixture to cool to ambient temperature overnight and then evaporated to an oil. Product was confirmed with 13 C and 1 H NMR and LCMS.
  • reaction mixture was purified by normal phase flash chromatography using a gradient of chloroform/isopropanol containing 5% ammonium hydroxide and desired 2-((1-(bis(octadecyloxy)phosphoryl)-3-ethoxy-3-oxoethyl)(methyl)amino)acetic acid obtained.
  • the title compound may be prepared by following substantially the procedure of Example 23 but substituting formyl phosphonic acid (Wagenknecht, J. H., Journal of the Electrochemical Society (1976), 123(5), 620-4, U.S. Pat. No. 4,568,432, and WO9850391) for ethyl glyoxylate in similar proportions.

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IL231215A0 (en) 2014-04-30
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US20130066086A1 (en) 2013-03-14
US20150030541A1 (en) 2015-01-29
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